检测人血浆蛋白α_1-蛋白酶抑制剂活性的弹性蛋白酶动态显色法的优化及其验证  

Optimization and validation of kinetic chromogenic method for determination of α1-protease inhibitor activity in human plasma

作　　者：周雁翔[1] 周志军[1] 林连珍[1] 彭焱[1] 邹莉[1] 胡勇[1] 邹炜[1] 李策生[1] 

机构地区：[1]武汉生物制品研究所有限责任公司血液制剂研究室,湖北武汉430060

出　　处：《中国生物制品学杂志》2015年第4期406-410,共5页Chinese Journal of Biologicals

基　　金：国家高技术研究发展计划(863计划)(2013ACC001)

摘　　要：目的优化检测人血浆蛋白α1-蛋白酶抑制剂（α1-protease inhibitor,α1-PI）活性的弹性蛋白酶动态显色法,并进行验证。方法以微量滴定板为载体,将不同浓度的α1-PI与特定量的猪胰弹性蛋白酶（porcine pancreaselastase,PPE）混合,测定剩余的弹性蛋白酶催化特定底物的强度,判定样品中α1-PI的含量,通过比较不同浓度酶和底物的组合中标准曲线的线性范围,确定最佳反应条件。对优化的动态显色法进行准确度、精密度验证,并确定标准曲线的线性范围。采用优化后的方法对α1-PI制备工艺进行监控,并筛选冻干配方。结果 PPE与底物的浓度选择分别为0.2 U/ml和1.2 mg/ml。该方法检测α1-PI的线性范围在2-14μg/ml之间,r〉0.99。各浓度样品检测结果的CV均〈4%,回收率在99.4%-104-3%之间,准确性较好;同一样品由同一个实验员在同一试验中连续测定6次的CV为1.8%,同一样品由不同的实验员在不同日期内分别测定3次的CV为1.8%,重复性较好。纯化工艺过程中各样品比活均符合《欧洲药典》规定的〉0.7,总回收率为62.9%;筛选出可较好保护α1-PI活性的配方。结论优化后的弹性蛋白酶动态显色法可用于工艺样品中α1-PI活性的检测。Objective To optimize and validate the kinetic chromogenic method for determination of α1-protease inhibitor （PI） activity in human plasma. Methods The α1-PI at various concentrations were mixed with a given quantity of porcine pancreaselastase （ PPE ） using microtiter plate as a carrier, and the remaining PPE activity in catalysis of specified substrate was determined to judge the α1-PI content. The reaction condition was optimized by comparing the linear range of standard curve of combinations of various concentrations of chromogenic substrate and PPE. The optimized kinetic chromogenic assay was verified for accuracy and precision, of which the linear range of standard curve was determined. The process for preparation of α1-PI was monitored by the optimized kinetic chromogenie assay, based on which the formula for lyophilization was optimized. Results The optimal concentrations of PPE and substrate were 0. 2 U / ml and 1.2μg / ml respectively. The linear range of the method for determination of α1-PI was 2 -14μg / ml, with a r value of more than 0. 99. The CVs of determination results of samples at various concentrations were less than 4%, while the recovery rate was 99.4% - 104- 3%, indicating a high accuracy. The CV of six determination results of the same sample by one personnel in one test was 1. 8%, while that of three determination results of the same sample by various personnel on various working days was also 1.8%, indicating a high reproducibility. All the specific activities of samples in purification process were more than 0. 7, which met the requirements in European Pharmaceupia, while the total recovery rate was 62. 9%. The formula for lyophilization was optimized, by which the α1-PI activity was effectively maintained. Conclusion The optimized kinetic cbromogenic assay may be used for the determination of α1-PI activity in process samples.

关 键 词：动态显色法 α1-蛋白酶抑制剂 优化 验证 

分 类 号：O657.32[理学—分析化学]